Inhibition of excystation and metacystic development of Entamoeba invadens by the dinitroaniline herbicide oryzalin

The effect of oryzalin on excystation and metacystic development of Entamoeba invadens strain IP-1 was examined by transfer of cysts to a growth medium containing the drug. Excystation, which was assessed by counting the number of metacystic amoebae after induction of excystation, was inhibited by oryzalin in a concentration-dependent manner. Metacystic development, which was determined by the number of nuclei in metacystic amoebae, was also inhibited by oryzalin because the percentage of 4-nucleate amoebae at day 1 remained unchanged at day 3. The addition of oryzalin after the induction of excystation decreased the number of metacystic amoebae, compared with control cultures. When cysts were incubated for 1 day in growth medium plus oryzalin, little increase in the number of metacystic amoebae was observed after removal of the drug. Excystation and metacystic development were further inhibited when the cysts were incubated for 30 min in encystation medium containing oryzalin before transfer to growth medium with the drug. When cysts were incubated for 30 min in encystation medium before transfer to growth medium without the drug, metacystic amoebae decreased in number. Pretreatment of cysts with oryzalin for 30 min in phosphate-buffered saline markedly reduced viability and prevented excystation in growth medium with or without the drug. The results indicate that oryzalin inhibits excystation and metacystic development of E. invadens, suggesting that it may be an inhibitor of Entamoeba infection.     

Entamoeba invadens: inhibition of excystation and metacystic development by aphidicolin

The effect of aphidicolin, a specific inhibitor of the replicative DNA polymerases, on the excystation and metacystic development of Entamoeba invadens was examined. The protein profile of metacystic amoebae and their immunogenicity in the presence and absence of aphidicolin were also examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Excystation, which was assessed by counting the number of metacystic amoebae after the induction of excystation, was inhibited by aphidicolin in a concentration-dependent manner during incubation compared to the controls. Metacystic development, when determined by the number of nuclei in amoeba, was also inhibited by aphidicolin, because the percentage of 4-nucleate amoebae in cultures with aphidicolin during incubation was higher than that in cultures without the drug. The addition of aphidicolin to cultures at day 1 of incubation reduced the number of metacystic amoebae thereafter compared to cultures without the drug. The inhibitory effect of aphidicolin on excystation and metacystic development was reversed by removal of the drug. Pretreatment of cysts with aphidicolin before transfer to a growth medium containing the drug had no further effect on the excystation and metacystic development. Cellular proteins of metacystic amoebae with 4 nuclei, which were predominant even at day 3 in the cultures with aphidicolin, reacted strongly with rabbit anticyst serum absorbed with trophozoite proteins. In contrast, those of metacystic amoebae with 1 nucleus, which were predominant at day 3 in cultures without aphidicolin, no longer reacted with the absorbed anticyst serum, suggesting change in the expression of proteins during metacystic development.     

Entamoeba invadens: cysteine protease inhibitors block excystation and metacystic development

We examined the effects of six cysteine protease inhibitors on the excystation and metacystic development of Entamoeba invadens. Excystation, which was assessed by counting the number of metacystic amoebae after the induction of excystation, was inhibited by the cysteine protease inhibitors Z-Phe-Ala-DMK and E-64d in a concentration-dependent manner during incubation compared to the controls. Neither inhibitor had a significant effect on cyst viability; thus, their inhibitory effects were not due to the toxic effect on cysts. Metacystic development, when determined by the number of nuclei in amoeba, was also inhibited by these protease inhibitors, because the percentage of 4-nucleate amoebae was higher than in the controls on Day 3 of incubation. Although other cysteine protease inhibitors, Z-Phe-Phe-DMK, E-64, ALLM, and cathepsin inhibitor III, had a weak or little effect on the excystation, they inhibited cysteine protease activity in the lysates of E. invadens cysts. Broad bands with gelatinase activity of metacystic amoebae, as well as cysts and trophozoites, were detected in the gelatin substrate gel electrophores and were inhibited by Z-Phe-Ala-DMK. There was a difference in the protease composition between cysts and trophozoites, and the protease composition of metacystic amoebae changed from cyst-type to trophozoite-type during development. These results strongly suggest that cysteine proteases contribute to the excystation and metacystic development of E. invadens, which leads to successful infection.     

Effect of jasplakinolide on the growth, encystation, and actin cytoskeleton of Entamoeba histolytica and Entamoeba invadens

The effect of jasplakinolide. an actin-polymerizing and filament-stabilizing drug, on the growth, encystation, and actin cytoskeleton of Entamoeba histolytica and Entamoeba invadens was examined. Jasplakinolide inhibited the growth of E. histolytica strain HM-1:IMSS and E. invadens strain IP-1 in a concentration-dependent manner, the latter being more resistant to the drug. The inhibitory effect of jasplakinolide on the growth of E. histolytica trophozoites was reversed by removal of the drug after exposure to 1 microM for 1 day. Encystation of E. invadens as induced in vitro was also inhibited by jasplakinolide. Trophozoites exposed to jasplakinolide in encystation medium for 1 day did not encyst after removal of the drug, whereas those exposed to the drug in growth medium for 7 days did encyst without the drug. The process of cyst maturation was unaffected by jasplakinolide. Large round structures were formed in trophozoites of both amoebae grown with jasplakinolide; these were identified as F-actin aggregates by staining with fluorescent phalloidin. Accumulation in trophozoites of both amoebae of actin aggregates was observed after culture in jasplakinolide. Also, E. invadens cysts formed from trophozoites treated with jasplakinolide contained the actin aggregate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis revealed that the jasplakinolide treatment led to an increase in the proportion of F-actin associated with formation of the aggregate. The results suggest that aggregates are formed from the cortical flow of F-actin filaments, and that these filaments would normally be depolymerized but are artificially stabilized by jasplakinolide binding.     

Entamoeba invadens: Identification of ADF/cofilin and their expression analysis in relation to encystation and excystation

The differentiation processes of excystation and encystation of Entamoeba are essential for infection and completion of their life-cycle, and the processes need cell motility and its control by actin cytoskeletal reorganization. This study investigated actin depolymerizing factor (ADF)/cofilin (Cfl) family proteins, which are important molecules in actin cytoskeletal reorganization, in Entamoeba invadens in relation to the encystation and excystation. Axenic culture systems were used to induce encystation and excystation. A homology search of the E. invadens genome database and molecular cloning identified three ADF/Cfl family proteins of the parasite (named for short as EiCfl-1, EiCfl-2, and EiCfl-3). This is different from other Entamoeba species, i.e. Entamoeba histolytica and Entamoeba dispar, each of which has only one ADF/Cfl family protein. These ADF/Cfl of E. invadens do not have Ser3 (serine locates third from first methionine), similar to E. histolytica, E. dispar, Saccharomyces cerevisiae and Schizosaccharomyces pombe, although the activity of ADF/Cfl is negatively regulated by phosphorylation of the Ser3 in metazoans. Phylogenetic analysis revealed that Entamoeba Cfl formed a distinctive clade that is separate from other organisms, and the branches of the tree were separated in two consistent with the presence and absence of Ser3. Rabbit anti-EiCfl-2 serum reacted with all recombinant EiCfls and EiCfl in lysates of cysts, trophozoites and metacystic amoebae. Immunofluorescence staining with this antiserum showed co-localization of EiCfl with actin beneath the cell membrane through the life stages. Both proteins proved to be rich in pseudopodia of trophozoites and metacystic amoebae. Real-time RT-PCR showed that mRNAs of EiCfl-2 and actins were highly expressed, but there were few mRNA of EiCfl-1 and EiCfl-3. Remarkably decreased mRNA levels were observed in EiCfl-2 and actins during encystation. All three EiCfls and actins became transcribed after the induction of excystation. The mRNAs of only EiCfl-1 and EiCfl-3 increased remarkably when the excystation was induced in the presence of cytochalasin D. These findings demonstrate that EiCfl-2 and actins co-localize beneath the cell membrane in trophozoites and cysts as well as metacystic amoebae being rich in pseudopodia, that EiCfl-1 and EiCfl-3 are expressed only after the induction of excystation, and that enhanced excystation by cytochalasin D is associated with high expression of EiCfl-1 and EiCfl-3.     

Involvement of calcium signaling and the actin cytoskeleton in the membrane block to polyspermy in mouse eggs

This study examines the effects of actin microfilament-disrupting drugs on events of fertilization, with emphasis on gamete membrane interactions. Mouse eggs, freed of their zonae pellucidae, were treated with drugs that perturb the actin cytoskeleton by different mechanisms (cytochalasin B, cytochalasin D, jasplakinolide, latrunculin B) and then inseminated. Cytochalasin B, jasplakinolide, and latrunculin B treatments resulted in a decrease in the percentage of eggs fertilized and the average number of sperm fused per egg. However, cytochalasin D treatment resulted in an increase in the average number of sperm fused per egg and the percentage of polyspermic eggs. This increase in polyspermy occurred despite the observation that cytochalasin D treatment caused a decrease in sperm-egg binding and did not affect spontaneous acrosome reactions or sperm motility. This suggested that cytochalasin D-treated eggs had an impaired ability to establish a block to polyspermy at the level of the plasma membrane. The effect of cytochalasin D on the block to polyspermy was not due to a general disruption of egg activation because sperm-induced calcium oscillations and cortical granule exocytosis were similar in cytochalasin D-treated and control eggs. However, buffering of intracellular calcium levels with the calcium chelator BAPTA-AM resulted in an increase in polyspermy. Together, these data suggest that a postfertilization decrease in egg membrane receptivity to sperm requires functions of the egg actin cytoskeleton that are disrupted by cytochalasin D. Furthermore, egg activation-associated increased intracellular calcium levels are necessary but not sufficient to affect postfertilization membrane dynamics that contribute to a membrane block to polyspermy.     

Latrunculins--novel marine macrolides that disrupt microfilament organization and affect cell growth: I. Comparison with cytochalasin D

The latrunculins are architecturally novel marine compounds isolated from the Red Sea sponge Latrunculia magnifica. In vivo, they alter cell shape, disrupt microfilament organization, and inhibit the microfilament-mediated processes of fertilization and early development. In vitro, latrunculin A was recently found to affect the polymerization of pure actin in a manner consistent with the formation of a 1:1 molar complex with G-actin. These in vitro effects as well as previous indications that the latrunculins are more potent than the cytochalasins suggest differences in the in vivo mode of action of the two classes of drugs. To elucidate these differences we have compared the short- and long-term effects of latrunculins on cell shape and actin organization to those of cytochalasin D. Exposure of hamster fibroblast NIL8 cells for 1-3 hr to latrunculin A, latrunculin B, and cytochalasin D causes concentration-dependent changes in cell shape and actin organization. However, the latrunculin-induced changes were strikingly different from those induced by cytochalasin D. Furthermore, while initial effects were manifest with both latrunculin A and cytochalasin D already at concentrations of about 0.03 microgram/ml, latrunculin A caused complete rounding up of all cells at 0.2 microgram/ml, whereas with cytochalasin D maximum contraction was reached at concentrations 10-20 times higher. The short-term effects of latrunculin B were similar to those of latrunculin A although latrunculin B was slightly less potent. All three drugs inhibited cytokinesis in synchronized cells, but their long-term effects were markedly different. NIL8 cells treated with latrunculin A maintained their altered state for extended periods. In contrast, the effects of cytochalasin D progressed with time in culture, and the latrunculin B-induced changes were transient in the continued presence of the drug. These transient effects were found to be due to a gradual inactivation of latrunculin B by serum and were used to compare recovery patterns of cell shape and actin organization in two different cell lines. This comparison showed that the transient effects of latrunculin B were fully reversible for the NIL8 cells and not for the mouse neuroblastoma N1E-115 cells.     

Actin assembly plays a variable, but not obligatory role in receptor-mediated endocytosis in mammalian cells

Three cell-permeant compounds, cytochalasin D, latrunculin A and jasplakinolide, which perturb intracellular actin dynamics by distinct mechanisms, were used to probe the role of filamentous actin and actin assembly in clathrin-mediated endocytosis in mammalian cells. These compounds had variable effects on receptor-mediated endocytosis of transferrin that depended on both the cell line and the experimental protocol employed. Endocytosis in A431 cells assayed in suspension was inhibited by latrunculin A and jasplakinolide, but resistant to cytochalasin D, whereas neither compound inhibited endocytosis in adherent A431 cells. In contrast, endocytosis in adherent CHO cells was more sensitive to disruption of the actin cytoskeleton than endocytosis in CHO cells grown or assayed in suspension. Endocytosis in other cell types, including nonadherent K562 human erythroleukemic cells or adherent Cos-7 cells was unaffected by disruption of the actin cytoskeleton. While it remains possible that actin filaments can play an accessory role in receptor-mediated endocytosis, these discordant results indicate that actin assembly does not play an obligatory role in endocytic coated vesicle formation in cultured mammalian cells.     

The platelet cytoskeleton regulates the affinity of the integrin alpha(IIb)beta(3) for fibrinogen.

Agonist-generated inside-out signals enable the platelet integrin alpha(IIb)beta(3) to bind soluble ligands such as fibrinogen. We found that inhibiting actin polymerization in unstimulated platelets with cytochalasin D or latrunculin A mimics the effects of platelet agonists by inducing fibrinogen binding to alpha(IIb)beta(3). By contrast, stabilizing actin filaments with jasplakinolide prevented cytochalasin D-, latrunculin A-, and ADP-induced fibrinogen binding. Cytochalasin D- and latrunculin A-induced fibrinogen was inhibited by ADP scavengers, suggesting that subthreshold concentrations of ADP provided the stimulus for the actin filament turnover required to see cytochalasin D and latrunculin A effects. Gelsolin, which severs actin filaments, is activated by calcium, whereas the actin disassembly factor cofilin is inhibited by serine phosphorylation. Consistent with a role for these factors in regulating alpha(IIb)beta(3) function, cytochalasin D- and latrunculin A-induced fibrinogen binding was inhibited by the intracellular calcium chelators 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid acetoxymethyl ester and EGTA acetoxymethyl ester and the Ser/Thr phosphatase inhibitors okadaic acid and calyculin A. Our results suggest that the actin cytoskeleton in unstimulated platelets constrains alpha(IIb)beta(3) in a low affinity state. We propose that agonist-stimulated increases in platelet cytosolic calcium initiate actin filament turnover. Increased actin filament turnover then relieves cytoskeletal constraints on alpha(IIb)beta(3), allowing it to assume the high affinity conformation required for soluble ligand binding.     

Actin Turnover-Mediated Gravity Response in Maize Root Apices: Gravitropism of Decapped Roots Implicates Gravisensing Outside of the Root Cap

The dynamic actin cytoskeleton has been proposed to be linked to gravity sensing in plants but the mechanistic understanding of these processes remains unknown. We have performed detailed pharmacological analyses of the role of the dynamic actin cytoskeleton in gravibending of maize (Zea mays) root apices. Depolymerization of actin filaments with two drugs having different mode of their actions, cytochalasin D and latrunculin B, stimulated root gravibending. By contrast, drug-induced stimulation of actin polymerization and inhibition of actin turnover, using two different agents phalloidin and jasplakinolide, compromised the root gravibending. Importantly, all these actin drugs inhibited root growth to similar extents suggesting that high actin turnover is essential for the gravity-related growth responses rather than for the general growth process. Both latrunculin B and cytochalasin D treatments inhibited root growth but restored gravibending of the decapped root apices, indicating that there is a strong potential for effective actin-mediated gravity sensing outside the cap. This elusive gravity sensing outside the root cap is dependent not only on the high rate of actin turnover but also on weakening of myosin activities, as general inhibition of myosin ATPases induced stimulation of gravibending of the decapped root apices. Collectively, these data provide evidence for the actin turnover-mediated gravity sensing outside the root cap.Key Words: actin cytoskeleton, gravisensing, graviresponding, root cap     

Modulating the Actin Cytoskeleton Affects Mechanically Induced Signal Transduction and Differentiation in Mesenchymal Stem Cells

Mechanical interactions of mesenchymal stem cells (MSC) with the environment play a significant role in controlling the diverse biological functions of these cells. Mechanical forces are transduced by integrins to the actin cytoskeleton that functions as a scaffold to switch mechanical signals into biochemical pathways. To explore the significance of cytoskeletal mechanisms in human MSC we modulated the actin cytoskeleton using the depolymerising drugs cytochalasin D (CytD) and latrunculin A (LatA), as well as the stabilizing drug jasplakinolide (Jasp) and examined the activation of the signalling molecules ERK and AKT during mechanical loading. All three drugs provoked significant changes in cell morphology and organisation of the cytoskeleton. Application of mechanical forces to β1-integrin receptors using magnetic beads without deformation of the cell shape induced a phosphorylation of ERK and AKT. Of the two drugs that inhibited the cytoskeletal polymerization, LatA completely blocked the activation of ERK and AKT due to mechanical forces, whereas CytD inhibited the activation of AKT but not of ERK. Activation of both signalling molecules by integrin loading was not affected due to cell treatment with the cytoskeleton stabilizing drug Jasp. To correlate the effects of the drugs on mechanically induced activation of AKT and ERK with parameters of MSC differentiation, we studied ALP activity as a marker for osteogenic differentiation and examined the uptake of fat droplets as marker for adipogenic differentiation in the presence of the drugs. All three drugs inhibited ALP activity of MSC in osteogenic differentiation medium. Adipogenic differentiation was enhanced by CytD and Jasp, but not by LatA. The results indicate that modulation of the cytoskeleton using perturbing drugs can differentially modify both mechanically induced signal transduction and MSC differentiation. In addition to activation of the signalling molecules ERK and AKT, other cytoskeletal mechanisms are involved in MSC differentiation.     

Induction of anti-actin drug resistance in Tetrahymena

Both cytochalasin D and latrunculin B reversibly inhibited Tetrahymena phagocytosis at concentrations similar to those effective in mammalian systems, even though ciliate actins are known to be highly divergent from mammalian actins. Overnight exposure to relatively low (0.25 microM) concentrations of latrunculin B induced resistance in Tetrahymena to the inhibitory effects of that drug, as well as cross-resistance to cytochalasin D. However, much higher (> 30 microM) concentrations of cytochalasin D were required for induction of cross-resistance to latrunculin B. Anti-actin drug resistance in Tetrahymena may involve a general multidrug resistance mechanism and/or specific feedback regulation of F-actin assembly and stability.     

Actin stress fiber retraction and aggresome formation is a common cellular response to actin toxins

F-actin-stabilizing drugs induce actin aggresome formation. In this study, we found that an actin-depolymerizing drug, latrunculin A (LatA), induced actin aggresomes. Actin stress fibers were retracted and disappeared in minutes, but a large aggresome formed in consequence of LatA treatment. Because cytochalasin D and mycalolide also induced aggresome formation, these results suggest that actin aggresome formation is a common cellular response to actin toxins.     

Actin Stress Fiber Retraction and Aggresome Formation Is a Common Cellular Response to Actin Toxins

F-actin-stabilizing drugs induce actin aggresome formation. In this study, we found that an actin-depolymerizing drug, latrunculin A (LatA), induced actin aggresomes. Actin stress fibers were retracted and disappeared in minutes, but a large aggresome formed in consequence of LatA treatment. Because cytochalasin D and mycalolide also induced aggresome formation, these results suggest that actin aggresome formation is a common cellular response to actin toxins.     

PKC-dependent stimulation of EAAT3 glutamate transporter does not require the integrity of actin cytoskeleton

The activity and the membrane expression of EAAT3 glutamate transporter are stimulated upon PKC activation by phorbol esters in C6 rat glioma cells. To investigate the role of cytoskeleton in these effects, we have employed actin-perturbing toxins and found that the perturbation of actin cytoskeleton inhibits basal but not phorbol-stimulated EAAT3 activity and membrane trafficking. In the absence of phorbols, latrunculin A, a toxin that disassembles actin cytoskeleton, produced a rapid inhibition of EAAT3 activity, due to a decrease in transport V(max). The inhibitory effect was fully reversible and was not detected for other sodium dependent transport systems for amino acids. However, latrunculin did not prevent the increase in transport caused by phorbol esters and, moreover, cells pre-treated with phorbols were resistant to the inhibitory effect of the toxin on EAAT3 activity. Biotinylation experiments indicated that the inhibitory effect of latrunculin was attributable to a decreased expression of the carrier on the membrane, while the toxin did not suppress the PKC-dependent increase in EAAT3 membrane abundance. Latrunculin A effects on EAAT3 were shared by cytochalasin D, a toxin that disorganizes actin filaments with a distinct mechanism of action. On the contrary, a small, but significant, increase of EAAT3 activity was observed upon incubation with jasplakinolide, a drug that stabilizes actin microfilaments. Also jasplakinolide, however, did not hinder phorbol-dependent stimulation of aspartate transport. Colchicine, a toxin that disrupts microtubules, also lowered EAAT3 activity without preventing transport stimulation by phorbols, while microtubule stabilization by paclitaxel led to an increase in aspartate transport. It is concluded that, in C6 cells, the PKC-mediated stimulatory effects on EAAT3 are cytoskeleton-independent, while in the absence of phorbols, the transporter is partially inhibited by the disorganization of either actin microfilaments or microtubules. These results suggest that EAAT3 trafficking in C6 cells involves different pools of transporters.     

Jasplakinolide, an actin stabilizing agent, alters anaphase chromosome movements in crane-fly spermatocytes

We added jasplakinolide to anaphase crane-fly spermatocytes and determined its effects on chromosome movement. Previous work showed that the actin depolymerizing agents cytochalasin D or latrunculin B blocked or slowed chromosome movements. We studied the effects of jasplakinolide, a compound that stabilizes actin filaments. Jasplakinolide had the same effect on movements of each half- bivalent in a separating pair of half-bivalents, but different half-bivalent pairs in the same cell often responded differently, even when the concentrations of jasplakinolide varied by a factor of two. Jasplakinolide had no effect on about 20% of the pairs, but otherwise caused movements to slow, or to stop, or, rarely, to accelerate. When cells were kept in jasplakinolide, stopped pairs eventually resumed movement; slowed pairs did not change their speeds. Confocal microscopy indicated that neither the distributions of spindle actin filaments nor the distributions of spindle microtubules were altered by the jasplakinolide. It is possible that jasplakinolide binds to spindle actin and blocks critical binding sites, but we suggest that jasplakinolide affects anaphase chromosome movement by preventing actin-filament depolymerization that is necessary for anaphase to proceed. Overall, our data indicate that actin is involved in one of the redundant mechanisms cells use to move chromosomes.     

Tropomyosin isoforms define distinct microfilament populations with different drug susceptibility

Two tropomyosin isoforms, human Tm5(NM1) and Tm3, were over-expressed in B35 rat neuro-epithelial cells to examine preferential associations between specific actin and tropomyosin isoforms and to determine the role tropomyosin isoforms play in regulating the drug susceptibility of actin filament populations. Immunofluorescence staining and Western blot analysis were used to study the organisation of specific filament populations and their response to treatment with two widely used actin-destabilising drugs, latrunculin A and cytochalasin D. In Tm5(NM1) cells, we observed large stress fibres which showed predominant co-localisation of beta-actin and low-molecular-weight gamma-tropomyosin isoforms. Tm3 cells had an abundance of cellular protrusions which contained both the beta- and gamma-actin isoforms, predominately populated by high-molecular-weight alpha- and beta-tropomyosin isoforms. The stress fibres observed in Tm5(NM1) cells were more resistant to both latrunculin A and cytochalasin D than filaments containing the high-molecular-weight tropomyosins observed in Tm3 cells. Knockdown of the over-expressed Tm5(NM1) isoform with a human-specific Tm5(NM1) siRNA reversed the phenotype and caused a reversal in the observed drug resistance. We conclude that there are preferential associations between specific actin and tropomyosin isoforms, which are cell type specific, but it is the tropomyosin composition of a filament population which determines the susceptibility to actin-targeting drugs.     

Involvement of the mitogen-activated protein kinase SIMK in regulation of root hair tip growth

Mitogen-activated protein kinases (MAPKs) are involved in stress signaling to the actin cytoskeleton in yeast and animals. We have analyzed the function of the stress-activated alfalfa MAP kinase SIMK in root hairs. In epidermal cells, SIMK is predominantly nuclear. During root hair formation, SIMK was activated and redistributed from the nucleus into growing tips of root hairs possessing dense F-actin meshworks. Actin depolymerization by latrunculin B resulted in SIMK relocation to the nucleus. Conversely, upon actin stabilization with jasplakinolide, SIMK co-localized with thick actin cables in the cytoplasm. Importantly, latrunculin B and jasplakinolide were both found to activate SIMK in a root-derived cell culture. Loss of tip-focused SIMK and actin was induced by the MAPK kinase inhibitor UO 126 and resulted in aberrant root hairs. UO 126 inhibited targeted vesicle trafficking and polarized growth of root hairs. In contrast, overexpression of gain-of-function SIMK induced rapid tip growth of root hairs and could bypass growth inhibition by UO 126. These data indicate that SIMK plays a crucial role in root hair tip growth.     

In Vitro Excystation of Spironucleus muris

ABSTRACT. In vitro excystation of Spironucleus muris cysts, purified by sequential sucrose and Percoll gradients from mouse feces, was studied. Three in vitro excystation procedures, used for Giardia , were assessed to determine the most useful method. Excystation was monitored by light microscopy and subsequently characterized by transmission and scanning electron microscopy. Spironucleus muris excysted routinely at a level greater than 90% when induced in Hanks' balanced salt solution containing sodium bicarbonate at pH 2.0 and transferred to Tyrodes' salt solution as an excystation medium. Similarly, high rates of excystation were recorded after induction of S. muris cysts in 0.1 M potassium phosphate buffer (pH 7.0) with sodium bicarbonate and excystation in trypticase-yeast extract-iron medium (TYI medium) or phosphate-buffered saline. A lower rate and percentage of excystation were observed after induction of S. muris cysts in an aqueous hydrochloric acid solution (pH 2.0) followed by excystation in TYI medium. All excystation methods produced extremely active S. muris trophozoites with normal morphology. Nonexcysting S. muris cysts have a wall composed of an outer fibrous and an inner membranous portion. Following induction, numerous vesicles appeared in the peritrophic space. Excystation began by the cyst wall opening at one pole, and the anterior part of the trophozoite protruding from the cyst wall. The trophozoite emerged progressively from the cyst wall and the empty cyst wall appeared to collapse. Excysted trophozoites exhibited normal morphological features of S. muris trophozoites isolated from the mouse intestine.     

Cell relaxation after electrodeformation: effect of latrunculin A on cytoskeletal actin

Precise measurement of the mechanical properties of a cell provides useful information about its structural organization and physiological state. It is interesting to understand the effect of individual components on the mechanical properties of the entire cell. In this study, we investigate the influence of the cytoskeletal actin on the viscoelastic properties of a cell. Actin-specific agents, including latrunculin A and jasplakinolide, are used to alter the organization of the cytoskeletal actin. Brassica oleracea protoplasts are treated with the drugs and deformed under an external electric potential. The relaxation processes of single protoplasts after electrodeformation are measured. The data are analyzed by a model-independent spectrum recovery algorithm. Two distinct characteristic time constants are obtained from the relaxation spectra. Treatment with latrunculin A increases both of the relaxation time constants. The longest relaxation times for control, latrunculin A treated, and jasplakinolide treated cells are determined to be 0.28, 1.0, and 0.21 s, respectively.